Noninvasive prenatal diagnosis of Hb Bart's hydrops fetus using cell-free fetal DNA in maternal plasma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore a new noninvasive method for Hb Bart' s hydrops fetus by using PCR amplification efficiency discrimination between cell-free fetal DNA (cffDNA) and cell-free maternal DNA in maternal plasma.</p><p><b>METHODS</b>CffDNA samples from pregnant women bearing possible Hb Bart's hydrops fetus were collected. Fluorescent PCR and capillary electrophoresis (CE) were performed. Hb Bart's hydrops fetus was conclusively identified by different peak area ratio of products.</p><p><b>RESULTS</b>The peak area ratio of 30 cffDNA samples from Hb Bart' s hydrops fetus was much less than 1. However, the ratio of cffDNA sample from hydrops fetus due to other reasons was approximately equal to 1.</p><p><b>CONCLUSION</b>By using cffDNA fluorescent PCR and CE, a prenatal screening method for Hb Bart' s hydrops fetus was developed.</p>

A study on the (CA)n in FVIII gene in Han ethnic group in Guangxi Zhuang Autonomous Region by amplification polymorphisms combined with silver staining / 中华儿科杂志

<p><b>OBJECTIVE</b>Hemophilia A is an inherited bleeding disorder caused by defects in factor VIII (FVIII) gene. In the present study, the frequencies of the microsatellite alleles at introns 13 and 22 in the factor VIII gene were analyzed in the group of Han nationality in Guangxi Zhuang Autonomous Region to explore their diagnostic value for hemophilia A. These two sites were also used to detect the carriers in 13 hemophilia A families.</p><p><b>METHODS</b>Ninty-one individuals of Han ethnic group in Guangxi Zhuang Autonomous Region (135 X chromosomes) and 13 HA families were subjected to molecular studies. First, these two fragments were PCR amplified simultaneously. Then, silver staining was used later to show their polymorphisms. The investigators selected one sample at random to obtain its lengths of the PCR products at these two sites by ABI310 PCR amplifier. After counting its repeated numbers of (CA) according to the documents concerned, the repeated numbers of the other samples could be counted easily.</p><p><b>RESULTS</b>In the 91 individuals, 6 and 4 alleles were detected at these two sites, respectively. At intron 13 the allele frequencies ranged from 0.0002 to 0.5408 and polymorphism information content (PIC) was 0.5899. At intron 22 the allele frequencies ranged from 0.0444 to 0.4963 and its PIC was 0.5359. The actual heterozygosity for intron 13 and intron 22 were 0.6364 (28/44) and 0.5227 (23/44), respectively. In 13 hemophilia A families with positive history, 9 of them were diagnosed by this method and the diagnosis rate was 69%.</p><p><b>CONCLUSION</b>With high PICs, (CA)n at intron 13 and intron 22 were two valuable sites in the diagnosis of hemophilia A in the population of Han ethnic group in Guangxi Zhuang Autonomous Region. Compared with some other HA restrictive fragment length polymorphisms (RFLP), intron 22 (GT)n (AG)n was more informative.</p>

Identification of fetal nucleated erythrocytes in maternal blood using short tandem repeat typing after primer extension preamplification / 中华血液学杂志

<p><b>OBJECTIVE</b>To develop a method for identifying fetal nucleated erythrocytes (NRBCs) in maternal blood.</p><p><b>METHODS</b>NRBCs in maternal blood were detected by benzidine staining and collected by micromanipulation. After primer extension preamplification (PEP) of the entire genome from a single NRBC, short tandem repeat (STR) genotype was analysed after further amplification of this gene. Single NRBC was differentiated as fetal or maternal origin by comparison of STR genotype of NRBC with its corresponding parents.</p><p><b>RESULTS</b>NRBCs were found in all of 28 pregnant women in a range of 4 to 13 per 5 ml venous blood. About 43. 6% of NRBCs were determined to be fetal origin by STR typing.</p><p><b>CONCLUSION</b>This method provides effective identification of fetal NRBCs and allows non-invasive prenatal genetic diagnosis using single fetal NRBC.</p>

Significance of Serum Transferrin Receptors and Iron Overload in Children with Hemoglobin H Disease / 实用儿科临床杂志

Objective To measure the levels of serum transferrin receptors (sTfR) and serum ferritin (SF) in children with hemoglobin H disease for investigation the iron metabolism(sideropenic or iron overload) and to guide the proper intervention.To get a best index of judging the iron overload in HbH children by analysis of the values of sTfR and sTfR/lgSF.Methods Peripheral blood specimens were obtained from 50 cases of HbH children and 30 cases of healthy normal controls,the levels of sTfR and SF were examined by enzyme linked immunosorbent assays(ELISA).The diagnosis efficiency of sTfR and sTfR/lgSF in HbH children′s iron metabolism was judged by receiver operator cha-racteristic (ROC) curve analysis.Results The level of sTfR in HbH group was (6.74?1.02) mg?L-1,which was significantly lower than that in healthy control group[(8.09?0.67) mg?L-1](P

Detection of Fetal Sex Determining Region of Y Chromosome Gene Using Small Molecular Circulatory Cell-Free Fetal DNA in Maternal Plasma / 实用儿科临床杂志

Objective To evaluate the feasibility of cell free fetal DNA(cffDNA)-based noninvasive prenatal diagnosis,we developed a precise technique for fetal sex determining region of Y chromosome(SRY)gene detection using size-fractionated cell-free DNA in maternal plasma.Methods Peripheral blood samples were collected form 117 pregnant women.cffDNA was extracted based on a column absorbent method and isolated by agarose gel electrophoresis.A dulex-polymerase chain reaction(PCR)was used to detected SRY gene and glycerol-dehyde-phosphate dehydrogenase(GAPDH)gene.Results Both SRY and GAPDH gene were detected in 86 cffDNA samples from women bearing male fetuses.And only GAPDH gene was detected in 71 cffDNA samples from women bearing female fetuses.These results had a coincidence whit those of villus or amniotic fluid samples.The specificity and sensitivity reached 100%(117/117)and 100%(66/66),respectively.Conclusion By agarose gel electrophoresis,re-extratedand and dulex PCR,size-fractionated cell-free fetal DNA in maternal plasma can be selective enriched and used to noninvasive prenatal diagnosis of sex-linked disorders and single gene disorders.

Genetic and Clinical Features of Children with ?-Thalassemia Intermedia / 实用儿科临床杂志

Objective To analyze genetic and clinical features of 14 children with ?-thalassemia intermedia in Guangxi area.Methods ?-thalassemia genes,?-thalassemia genes,single nucleotide polymorphism(SNP) at position-158 of()~G?-globin gene,AT repeats polymorphisms of DNase I-hypersensitive site 2 of the ?-globin gene cluster locus control region(?-LCR-HS2) were detected by PCR techniques.Clinical data were analyzed.Results Genotype:1.Seven cases were homozygous or compound heterozygous for nt-28(A→G).Among them,2 cases′ genotypes were nt-28/nt-28,1 case was ?~E/ nt-28,2 cases were ?~0/nt-28,1 case(?~0/nt-28) co-inherited()~G?158(T) and 1 case(?~0/nt-28) co-inherited simultaneously()~G?-158(T) and——SEA ?-thalassemia-1 genes.2.Three cases with ?~0/?~0 presented()~G?-158(T),and other 3 cases co-inherited——SEA ?-thalassemia-1 genes.3.One patient with ?~0/?~0 co-inherited()~G?-158(T) and——SEA ?-thalassemia-1 genes.4.Six cases carrying()~G?-158(T) had(AT)_9 N_(12)(AT)_(10) sequences in ?-LCR-HS2.Phenotype:The values of Hb,MCV,HbF of 14 patients were(75.9?9.7) g/L,(68.9?5.9) fL,66.9%?16.3%,respectively.Except for 2 cases with genotypes of nt-28/nt-28 and 1 case with ?~E/nt-28 who had never been transfused,the others had more severe symptoms and required irregularly transfusion.Conclusions In the 14 children with ?-thalassemia intermedia from Guangxi area,nt-28(A→G),()~G?-158(T) and——SEA ?-thalassemia-1 gene are main alleviating gene factors.Incidence of(AT)_9 N_(12)(AT)_(10) sequence in ?-LCR-HS2 in these patients is high.Patients who are homozygous for nt-28 or compound heterozygous for ?~E have milder phenotypes.

Homozygous hemoglobin constant spring in Guangxi province / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the relationship between genotype and phenotype of homozygous hemoglobin Constant Spring (Hb CS) in Guangxi province, and to explore the reasons of missed diagnosis and the methods for screening and diagnosing.</p><p><b>METHODS</b>Screening Hb CS by acetate fibrous membrane electrophoresis with benzidine staining. Gene mutation of homozygous Hb CS by polymerase chain reaction (PCR) and DNA sequencing.</p><p><b>RESULTS</b>Out of the 9 patients, 4 had no clinical symptoms, 2 showed mild anemia and splenomegaly, 3 were jaundice. Hemoglobin levels were normal or mild anemia. MCVs were normal or reduced. Peripheral blood smear of all the patients displayed hypochromia, anisocytosis, poikilocytosis and target cells. The quantities of HbA(2) + Hb CS were 4.3% - 6.72%, while HbA(2) < 2%. Gene analysis confirmed the diagnosis of homozygous Hb CS.</p><p><b>CONCLUSION</b>There was quite different in clinical symptoms, hematological parameters and hemoglobin quantifications for patients with homozygous Hb CS. They might have no clinical and hematological signs and looked like the phenotype of Hb H disease. Homozygous Hb CS was very easy to miss diagnosis. Gene analysis can be helpful.</p>

Relationship between the expression of murine double minute 2 oncogene and non-Hodgkin lymphoma in childhood / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the expression of murine double minute 2 (MDM2) oncogene and non-Hodgkin lymphoma (NHL) in childhood.</p><p><b>METHODS</b>Thirty-one cases of NHL were enrolled in this study as patient group and 8 cases of lymphadenitis as control group. (1) Immunohistochemistry ultrasensitive S-P assay was used to detect the expression of MDM2 protein in pathological tissues in all cases. Positive cells were dyed yellow or brown in nuclei. MDM2 positive cell was defined as >/= 10% of the tumor cells were positive, which was overexpression of MDM2 protein. (2) RT-PCR (reverse transcription-polymerase chain reaction) was performed to value the overexpression of MDM2 mRNA in the pathological tissues and mononuclear cells in peripheral blood. While the ratio of MDM2/beta-actin was >16% was defined as overexpression of MDM2 mRNA.</p><p><b>RESULTS</b>(1) Rates of overexpression of MDM2 protein and MDM2 mRNA were 64.5% and 61.3%, respectively, which were significantly different as compared to that of control group (P < 0.05 and P < 0.01, respectively). (2) The relationship analysis among subgroups in the experiment group showed that the overexpression of MDM2 protein did not correlate with classifications of working formulation, cellular origin, sex, clinical stage and involved extranodal sites (P > 0.05), but significantly correlated with classifications of B status and the increased serum LDH level (P < 0.05). It was shown that the overexpression of MDM2 mRNA did not correlate with classifications of working formulation, cellular origin, sex and clinical stage (P > 0.05), significantly correlated with B status (P < 0.05), and was remarkably significantly correlated with the involved extranodal sites and the increased serum LDH level (P < 0.01). (3) It was demonstrated that the overexpression of MDM2 mRNA in the pathological tissues was similar to the overexpression of MDM2 protein in the pathological tissues and MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.655 and 0.571), and the overexpression of MDM2 protein in the pathological tissues was similar to that of MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.609).</p><p><b>CONCLUSIONS</b>(1) The rate of MDM2 oncogene overexpression was quite high. (2) The overexpression of MDM2 protein in pathological tissues determined by using immunohistochemistry ultrasensitive S-P assay was similar to that of MDM2 mRNA in pathological tissues detected by using RT-PCR method. Both methods might be used to detect the overexpression of MDM2 oncogene in the cases of childhood NHL. (3) The overexpression of MDM2 oncogene related to the poor status and poor prognosis of patients with childhood NHL.</p>

Beta-thalassemia mutations and single nucleotide polymorphism at -158 of Ggamma-globin gene associated with altered levels of Hb F in beta-thalassemia heterozygotes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of beta-thalassemia mutations and the single nucleotide polymorphism(SNP) at position -158 of (G)Gamma-globin gene to the altered levels of fetal hemoglobin(Hb F) of beta-thalassemia heterozygotes.</p><p><b>METHODS</b>Hb F was quantitated by alkali denaturation; beta-thalassemia mutations were determined by PCR-allelic specific oligonucleotide(PCR-ASO). The SNP at -158 was analyzed by amplification of (G)Gamma gene promoter fragments from the DNA, followed by Xmn I restriction enzyme digestion.</p><p><b>RESULTS</b>Among 63 cases with beta-thalassemia trait, 15 had Hb F levels above 2% (2.06%-10.44%). Six beta-thalassemia mutations were observed in this study, namely CD41/42(-TTCT), CD17(A-->T), nt28 (A-->G), CD71/72(+A), IVS-II-654(C-->T) and IVS-I-1(G-->T). There was no difference in the incidence of beta-thalassemia heterozygotes of CD41/42, CD17, CD71/72 and IVS-II-654 between 15 cases with Hb F>/=2% and 48 cases with Hb F<2%. Ten (15.9%) heterozygotes of (G)Gamma-158(C-->T)were detected among 63 cases, and 8 of them (53.33%) belonged to the group of Hb F>/=2% while the remaining 2 cases (4.17%) were in the group of Hb F<2%.</p><p><b>CONCLUSION</b>beta-thalassemia mutations of CD41/42, CD17, CD71/72, IVS-II-654 had no influence on Hb F levels, but (G)Gamma-158(C-->T) had a strong association with moderately increased Hb F levels in beta-thalassemia heterozygotes in the Guangxi area of China.</p>